Development of [18F]DASA-23 for Imaging Tumor Glycolysis Through Noninvasive Measurement of Pyruvate Kinase M2

Article abstract of DOI:10.1007/s11307-017-1068-8

Purpose: A hallmark of cancer is metabolic reprogramming, which is exploited by cancer cells to ensure rapid growth and survival. Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, a key step in tumor metabolism and growth. Recently, we reported the radiosynthesis of the first positron emission tomography tracer for visualizing PKM2 in vivo—i.e., [¹¹C]DASA-23. Due to the highly promising imaging results obtained with [¹¹C]DASA-23 in rodent model glioblastoma, we set out to generate an F-18-labeled version of this tracer, with the end goal of clinical translation in mind. Herein, we report the radiosynthesis of 1-((2-fluoro-6-[¹⁸F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([¹⁸F]DASA-23) and our initial investigation of its binding properties in cancer cells. Procedure: We synthesized [¹⁸F]DASA-23 via fluorination of 1-((2-fluoro-6-nitrophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine (10) with K[¹⁸F]F/K2.2.2 in N,N-dimethylformamide at 110?°C for 20?min. Subsequently, we evaluated uptake of [¹⁸F]DASA-23 in HeLa cervical adenocarcinoma cells and in vitro stability in human and mouse serum. Results: We successfully prepared [¹⁸F]DASA-23 in 2.61?±?1.54?% radiochemical yield (n?=?10, non-decay corrected at end of synthesis) with a specific activity of 2.59?±?0.44?Ci/μmol. Preliminary cell uptake experiments revealed high uptake in HeLa cells, which was effectively blocked by pretreating cells with the structurally distinct PKM2 activator, TEPP-46. [¹⁸F]DASA-23 remained intact in human and mouse serum up to 120?min. Conclusion: Herein, we have identified a F-18-labeled PKM2 specific radiotracer which shows potential for in vivo imaging. The promising cell uptake results reported herein warrant the further evaluation of [¹⁸F]DASA-23 for its ability to detect and monitor cancer noninvasively.

Full text of DOI:10.1007/s11307-017-1068-8

Mol Imaging Biol (2017)
DOI: 10.1007/s11307-017-1068-8
* World Molecular Imaging Society, 2017
RESEARCH ARTICLE
Development of [18F]DASA-23 for Imaging
Tumor Glycolysis Through Noninvasive
Measurement of Pyruvate Kinase M2
Corinne Beinat,1 Israt S. Alam,1 Michelle L. James,1,2 Ananth Srinivasan,1
Sanjiv Sam Gambhir1,3
1Department of Radiology, Molecular Imaging Program at Stanford, Stanford University, Stanford, CA, 943065, USA
2Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, 943065, USA
3Departments of Bioengineering and Materials Science and Engineering, Bio-X, Stanford University, Stanford, CA, 943065, USA
Abstract
Purpose: A hallmark of cancer is metabolic reprogramming, which is exploited by cancer cells to ensure
rapid growth and survival. Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, a key step in
tumor metabolism and growth. Recently, we reported the radiosynthesis of the first positron emission
tomography tracer for visualizing PKM2 in vivo—i.e., [11C]DASA-23. Due to the highly promising imaging
results obtained with [11C]DASA-23 in rodent model glioblastoma, we set out to generate an F-18-labeled
version of this tracer, with the end goal of clinical translation in mind. Herein, we report the radiosynthesis
of 1-((2-fluoro-6-[18F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([18F]DASA-23) and
our initial investigation of its binding properties in cancer cells.
Procedure: We synthesized [18F]DASA-23 via fluorination of 1-((2-fluoro-6-nitrophenyl)sulfonyl)-
4-((4-methoxyphenyl)sulfonyl)piperazine (10) with K[18F]F/K2.2.2 in N,N-dimethylformamide at
110 °C for 20 min. Subsequently, we evaluated uptake of [18F]DASA-23 in HeLa cervical
adenocarcinoma cells and in vitro stability in human and mouse serum.
Results: We successfully prepared [18F]DASA-23 in 2.61 1.54 % radiochemical yield (n = 10,
non-decay corrected at end of synthesis) with a specific activity of 2.59
0.44 Ci/μmol.
Preliminary cell uptake experiments revealed high uptake in HeLa cells, which was effectively
blocked by pretreating cells with the structurally distinct PKM2 activator, TEPP-46. [18F]DASA-
23 remained intact in human and mouse serum up to 120 min.
Conclusion: Herein, we have identified a F-18-labeled PKM2 specific radiotracer which shows
potential for in vivo imaging. The promising cell uptake results reported herein warrant the further
evaluation of [18F]DASA-23 for its ability to detect and monitor cancer noninvasively.
Key Words: [18F]DASA-23, Pyruvate kinase, Tumor glycolysis, Fluorine-18, Radiochemistry,
Positron emission tomography
to the metabolic needs of their unique biology has been a focus of
Introduction
In order to sustain rapid cell division and growth, tumors undergo
metabolic reprogramming. Understanding how cancer cells adjust
cancer research for many years [1]. The concept of metabolic
adaptation was first reported by Otto Warburg over 80 years ago,
after he observed that cancer cells exhibited enhanced consump-
tion of glucose and production of lactate in comparison to normal
tissue, even under aerobic conditions [2]. This phenomenon
became known as the Warburg effect. While normal,
Correspondence to: Sanjiv Gambhir; e-mail: sgambhir@stanford.edu

Beinat C. et al.: Development of [18F]DASA-23
differentiated cells maximize ATP production by mitochondrial
oxidative phosphorylation of glucose under normoxic conditions,
cancer cells produce substantially less ATP from glucose using
aerobic glycolysis [3]. The enhancement in aerobic glycolysis
combined with the dynamic processes in cancer cells enables
glycolytic intermediates to be redirected for the biosynthetic
production of cellular building blocks such as nucleotides, amino
acids, and lipids, while still producing ATP [3], therefore fulfilling
the requirements for macromolecular synthesis and energy
production [4, 5]. The Warburg effect has been extensively
exploited clinically to detect tumors and their response to
treatment by [18F]FDG. [18F]FDG PET noninvasively measures
rates of glucose metabolism and is approved for the diagnosis of
most cancers and has proven particularly useful as a staging and
restaging tool that can guide patient care [6, 7]. Since glucose
metabolism is an essential cellular process, [18F]FDG is not
specific for malignant cells. Particularly in the brain, which has a
high rate of glucose metabolism, and therefore a high physiolog-
ical uptake of [18F]FDG, it is extremely difficult to delineate brain
tumor margins with [18F]FDG PET imaging. There is a strong,
unmet need for PET imaging agents with selectivity for molecular
processes unique to cancer cells.
Pyruvate kinase (PK) catalyzes the final and rate-limiting
step in glycolysis, converting phosphoenol pyruvate (PEP)
to pyruvate by transferring the high-energy phosphate group
to adenosine diphosphate (ADP) and yielding ATP [3].
Reduced PK activity results in a diminished production of
pyruvate or prevention of the conversion of glucose to
pyruvate, therefore enabling the accumulation of upstream
glycolytic intermediates and shifting metabolism towards the
anabolic phase. Cancer cells exploit this effect by primarily
utilizing the PKM2 isoform of PK, whose activity can be
dynamically controlled between the less active PKM2 dimer
and the highly active PKM2 tetramer [8]. PKM2 is found in
most cells with the exception of adult muscle, brain, and
liver and is preferentially expressed in all cancers to date [8].
The dynamic equilibrium between the dimeric and tetra-
meric states of PKM2 enables proliferating tumor cells to
regulate their needs for anabolic and catabolic metabolism.
The alternative splicing of PKM2 is regulated by oncogenes
c-Myc and HIF-1 [9], with the quaternary structure of PKM2
regulated by the glycolytic intermediate fructose 1,6-
biphosphate (FBP) and growth factor signaling [10, 11].
The expression of PKM2 has been shown to be increased in
a diverse range of human cancers, including lung, breast,
prostate, blood, cervix, kidney, bladder, and colon, com-
pared to the matched normal tissues [12]. PKM2 expression
is linked to increased uptake of glucose, enhanced lactate
production, and a decrease in oxygen consumption, effects
which can be reversed by genetic modifications to replace
PKM2 expression with PKM1 [8, 13]. The Warburg effect is
therefore partly mediated by PKM2 expression, with the
high expression of dimeric PKM2 in cancer cells contribut-
ing to anabolic glucose metabolism, promoting macromo-
lecular biosynthesis and benefiting cancer cell proliferation
and growth [13].
In recent years, PKM2 has been explored as a potential
target for cancer therapy through the development of small
molecule activators that promote and stabilize active
tetramer formation [14, 15]. Multiple classes of PKM2
activators have been developed, and representative examples
are shown in Fig. 1. These examples include a series of N-N
′-diarylsulfonamides (1), thieno[3,2-b]pyrrole[3,2-
d]pyridazinones (2–3), 1-(sulfonyl)-5-(arylsulfonyl)indoline
(4), 2-((1H-benzo[d]imidazole-1-yl)methyl)-4H-pyrido[1,2-
a]pyrimidin-4-ones (5), 3-(trifluoromethyl)-1H-pyrazole-5-
carboxamide (6), and more recently 7-azaindole derivative
(7). Clinically relevant imaging agents for the direct,
noninvasive measurement of cancer-specific biomarkers are
a topic of extreme interest and importance, particularly for
brain tumors. Although [18F]FDG PET is approved for use
for the diagnosis of most cancers, a high background uptake
by surrounding healthy tissue can mask tumor uptake, for
example, in the normal brain [16]. Consequently, the
noninvasive measurement of PKM2 has the potential to
play an important role in the detection and management of
malignancies where [18F]FDG fails. To our knowledge,
there is currently no clinically approved PET tracer that can
be specifically used to visualize PKM2 expression. To
address this need, we have recently developed the PET
Fig. 1. Structures of representative PKM2 activators.

Beinat C. et al.: Development of [18F]DASA-23
tracer [1-((2,6-difluorophenyl)sulfon-yl)-4-(4-
([11C])methoxyphenyl)sulfonyl)piperazine] ([11C]DASA-23,
[
((4-methoxyphenyl)sulfonyl)piperazine (Scheme 1) was pre-
pared from 4-methoxybenzenesulfonyl chloride based on a
reported literature procedure [14]. Cold DASA-23 standard
was also prepared according to the same literature proce-
dure. High-performance liquid chromatography (HPLC)
grade acetonitrile (CH3CN) and Millipore 18 mΩ water
were used for [18F]DASA-23 purifications which were
performed on a Dionex Summit HPLC system (Dionex
Corporation, Sunnyvale, CA) equipped with a 340-U four-
channel UV-Vis absorbance detector and radioactivity
detector (Carroll & Ramsey Associates, model 105S,
Berkeley, CA). UV detection wavelengths were 254 and
280 nm for all the experiments. Semipreparative HPLC
reversed-phase column (Phenomenex Gemini, C18,
250 × 10 mm, 5 μM) was used for purification of
[18F]DASA-23. The mobile phase for the purification of
[18F]DASA-23 was water and acetonitrile. Radioactivity
measurements were performed by a CRC-15R PET dose
calibrator (Capintec Inc., Ramsey, NJ). Electron spray
ionization mass spectrometry was done by Vincent Coates
Foundation Mass Spectrometry Laboratory, Stanford Uni-
11C]1) [17]. We have demonstrated the ability of
[11C]DASA-23 to detect tumor-specific PKM2 in subcuta-
neous and orthotopic mouse models of human glioma
in vivo. These encouraging findings provided a foundation
for the clinical translation of [11C]DASA-23 for the imaging
of primary brain tumors and other tumors that metastasize to
the brain. Unfortunately, the physical characteristics of C-11
limit its clinical utility and practicality. The relatively short
half-life of C-11 (t1/2 = 20.4 min) requires an on-site
cyclotron within hospitals and imaging centers and conse-
quently hinders the clinical application of this diagnostic
radiotracer. The increasing clinical use of PET imaging and
the limited availability of cyclotrons within clinical PET
facilities have resulted in the development of numerous PET
radiotracers labeled with F-18. The half-life of F-18
(t1/2 = 110 min) potentially allows remote site synthesis
which has been extensively demonstrated with the world-
wide use and distribution of [18F]FDG. The presence of
fluorine atoms in the native structure of DASA-23 poses a
great challenge in order to introduce the F-18 label into its
native position with nucleophilic fluorination. An advantage
1
versity. H nuclear magnetic resonance (NMR) spectra were
taken on an Agilent 400 MHz spectrometer.
of radiolabeling at these native sites is that 1-((2-fluoro-6-
1 8
( f l u o r o -
F ) p h e n y l ) s u l f o n y l ) - 4 - ( ( 4 -
methoxyphenyl)sulfonyl)piperazine ([18F]DASA-23) will
maintain the same binding characteristics and specificity
towards PKM2 as [11C]DASA-23.
Radiochemistry
No carrier-added [18F]fluoride was prepared by the
18O(p,n)18F nuclear reaction on a GE PETtrace cyclotron.
[18F]Fluoride drying and synthesis of crude [18F]PKM2
were completed in the GE TRACERlab FX-FN automated
synthesis module.
Herein, we report a radiofluorination method for the
preparation of [18F]DASA-23. Additionally, we discuss the
stability of [18F]DASA-23 and its suitability for measuring
PKM2 expression in vitro.
Tert-Butyl 4-((4-
Materials and Methods
Methoxyphenyl)Sulfonyl)Piperazine-1-Carboxyl-
General
ate (8)
Chemicals were purchased from Aldrich Chemical Company
(Milwaukee, WI). 1-((2-Fluoro-6-nitrophenyl)sulfonyl)-4-
1-Boc-piperazine (500 mg, 2.68 mmol, 1 equiv.) was
suspended in dichloromethane (10 ml) and cooled (0 °C).
Scheme 1. Synthesis of nitro precursor 10. Reagents and conditions. a NEt3, CH2Cl2, 0 °C, 1 h, 42 %. b TFA, CH2Cl2, 0 °C,
1 h. c NEt3, CH2Cl2, 0 °C, 1 h, 85 %.

Beinat C. et al.: Development of [18F]DASA-23
Triethylamine (747 μl, 5.37 mmol, 2.0 equiv.) was added
followed by the addition of 4-methoxybenzenesulfonyl
chloride (609 mg, 2.95 mmol, 1.1 equiv.). The reaction
was maintained at 0 °C for 1 h and then quenched with
saturated aqueous ammonium chloride (~5 ml). The organic
layer was washed with saturated ammonium chloride
(2 × 10 ml), brine (10 ml), dried over sodium sulfate, and
concentrated under reduced pressure. The crude residue was
subsequently purified by flash chromatography on silica
eluting with hexane-ethyl acetate (65:35) to give tert-butyl 4-
((4-methoxyphenyl)sulfonyl)piperazine-1-carboxylate (8)
(396 mg, 42 % yield) as a colorless solid: δH (400 MHz,
CDCl3) = 7.68 (2 H, d, J 9.2 Hz), 7.0 (2 H, d, J 9.2 Hz), 3.86
(3 H, s), 3.90 (4 H, m), 2.94 (4 H, m).
completion after 1 h. The reaction was quenched with
saturated aqueous ammonium chloride (~5 ml). The organic
layer was washed twice with saturated aqueous ammonium
chloride, once with brine, dried over sodium sulfate,
concentrated in vacuo, and then dissolved in DMSO and
purified by reversed-phase HPLC to yield 1-((2,6-
d i f l u o r o p h e n y l ) s u l f o n y l ) - 4 - ( ( 4 -
methoxyphenyl)sulfonyl)piperazine (1, DASA-23) (152 mg,
77 %) as a colorless solid: δH (400 MHz, CDCl3) = 7.67 (2
H, d, J 8.8 Hz), 7.54 (1 H, m), 7.03 (4 H, m), 3.89 (3 H, s),
3.38 (4 H, m), 3.11 (4 H, m); m/z (ESI) 430 ([M + H]+, 100).
1-((2-Fluoro-6-(Fluoro-18F)Phenyl)Sulfonyl)-4-
((4-Methoxyphenyl)Sulfonyl)Piperazine
([18F]DASA-23, [18F]1)
1-((4-Methoxyphenyl)Sulfonyl)Piperazine (9)
No carrier-added aqueous [18F]fluoride ion was produced on
a PETtrace cyclotron (GE Healthcare, Sweden) by irradia-
tion of a 1.6 ml water target using a 16 MeV proton beam on
95 % enriched [18O]H2O by the [18O(p,n)18F] nuclear
reaction. [18F]Fluoride in [18O]H2O was transferred to a
GE TRACERlab FXFN synthesizer and trapped on an anion
exchange resin (QMA cartridge in carbonate form).
[18F]Fluoride ions were then eluted into the reactor using
an eluent solution containing 3.5 mg of K2CO3 and 15 mg of
Kryptofix 2.2.2 (K2.2.2: 4,7,13,16,21,24-hexaoxa-1,10-
diazabicyclo[8.8.8]hexacosan) in acetronitrile (0.9 ml) and
water (0.1 ml). The solution was then azeotropically dried at
65 °C under helium flow and vacuum, followed by heating
at 88 °C under vacuum. Nitro precursor (10) (300 μg) was
dissolved in N,N-dimethylformamide (DMF) (1 ml) and
added to the dry Kryptofix 2.2.2/K+[18F−] complex, and
mixture was allowed to react at 110 °C for 20 min. After
cooling to 40 °C, the reaction mixture was diluted with
sterile water (7 ml) and passed through a C18 Sep-Pak
cartridge. The radiolabeled product was subsequently eluted
from the C18 Sep-Pak with acetonitrile (1.5 ml) and sterile
water (1.5 ml). The resulting crude mixture was then
injected onto a HPLC Phenomenex Gemini C-18, 5-μm
(10 × 250 mm) semipreparative reversed-phase column. A
mobile phase of H2O (0.1 % TFA)/acetonitrile (0.1 % TFA)
was used with a flow rate of 3.0 ml/min, and the retention
time (tR) of [18F]1 was 17.8 min. The radioactive peak
corresponding to [18F]1 was diluted in a round-bottom flask
containing sterile water (20 ml) and then trapped on a C18
Sep-Pak. The trapped, purified radiolabeled product was
eluted from the C18 Sep-Pak using ethanol (1 ml) and saline
(9 ml). The formulated product was then transferred into a
sterile, pyrogen-free 30-ml vial. The radiochemical yield was
Tert-butyl 4-((4-methoxyphenyl)sulfonyl)piperazine-1-car-
boxylate (180 mg, 0.51 mmol) was dissolved in dichloro-
methane (3 ml) and cooled to 0 °C. Trifluoroacetic acid
(TFA) (1 ml) was added, and after 1 h, the reaction was
removed from the ice bath and the solvents removed in
v a c u o t o y i e l d t h e T F A s a l t o f 1 - ( ( 4 -
methoxyphenyl)sulfonyl)piperazine (9), which was used in
the next step without purification.
1-((2-Fluoro-6-Nitrophenyl)Sulfonyl)-4-((4-
Methoxyphenyl)Sulfonyl)Piperazine (10)
The oily residue (9) was dissolved in dichloromethane (5 ml)
and cooled to 0 °C. Triethylamine (258 μl, 1.85 mmol, 4
equiv.) was added followed by the addition of 2-fluoro-6-
nitrobenzenesulfonyl chloride (109 mg, 0.46 mmol, 1
equiv.). After 1 h, the reaction was quenched with addition
of saturated aqueous ammonium chloride (~5 ml). The
organic layer was washed with saturated aqueous ammonium
chloride (2 × 10 ml), brine (10 ml), dried over sodium
sulfate, and concentrated under reduced pressure. The crude
residue was dissolved in DMSO and purified by reversed-
phase HPLC to yield 1-((2-fluoro-6-nitrophenyl)sulfonyl)-4-
((4-methoxyphenyl)sulfonyl)piperazine (10) (180 mg, 85 %)
as a colorless solid: δH (400 MHz, CDCl3) = 7.67 (3 H, m),
7.40 (1 H, t, J 8.8 Hz), 7.33 (1 H, d, J 8.0 Hz), 7.01 (2 H, d,
J 8.8 Hz), 3.89 (3 H, s), 3.49 (4 H, m), 3.11 (4 H, m); m/z
(ESI) 459 ([M + H]+, 100).
1-((2,6-Difluorophenyl)Sulfonyl)-4-((4-
Methoxyphenyl)Sulfonyl)Piperazine (1, DASA-23)
2.61
1.54 % (non-decay corrected at end of synthesis
0.44 Ci/μmol
The oily residue (9) was dissolved in dichloromethane (5 ml)
and cooled to 0 °C. Triethylamine (258 μl, 1.85 mmol, 4
equiv.) was added followed by portionwise addition of 2,6-
difluoronitrobenzenesulfonyl chloride (109 mg, 0.46 mmol,
1 equiv.). The reaction was monitored by TLC and showed
(EOS)) with a specific activity of 2.59
(n = 10) and purity greater than 95 %. The chemical and
radiochemical purities were determined by reversed-phase
analytical HPLC Phenomenex Gemini C-18, 5 μm
(4.6 × 250 mm). A mobile phase of H2O (0.1 % TFA)/

Beinat C. et al.: Development of [18F]DASA-23
Scheme 2. Synthesis of DASA-23 cold standard. Reagents and conditions. a NEt3, CH2Cl2, 0 °C, 1 h, 77 %.
acetonitrile (0.1 % TFA) was used with a flow rate of 1.0 ml/
[18F]DASA-23 (130–150 μCi, 20 μl volume) was added to
min, and tR of [18F]1 was 10.3 min.
330 μl of supernatant. For control, the same volume of
radiolabeled compound in 330 μl PBS was used. After
vortexing the radiolabeled mixtures, 70 μl aliquots were
incubated at 37 °C. At predetermined time points (0, 15, 30,
60, and 120 min), 140 μl of ice-cold acetonitrile was added
to corresponding samples to stop metabolism. After
vortexing and centrifuging samples (10 min, 13,000 rpm),
the supernatants were analyzed by analytical HPLC using
the same conditions described above.
Cell Uptake Studies
HeLa cells (0.5 × 106) were plated into 6-well plates
overnight before [18F]DASA-23 uptake analysis. On the
day of the experiment, fresh, prewarmed DMEM containing
25 μCi of [18F]DASA-23 was added to individual wells
(1 ml per well). Cells were incubated with [18F]DASA-23 at
37 °C and 5 % CO2 over a 60-min time course. For blocking
studies, TEPP-46 (Cayman Chemical, 0.25 μM, at a final
concentration of 0.9 % DMSO) was added to Hela cells
straight after [18F]DASA-23 addition and cells were incu-
bated for 30 min. At the specified time points, incubation
plates were put on ice, washed with ice-cold phosphate-
buffered saline (PBS) (3 × 500 μl), and lysed in
radioimmunoprecipitation assay buffer (Thermo Fisher
Scientific Inc.; 500 μl). A portion of the cell lysates
(200 μl) were used to determine the amount of decay-
corrected radioactivity on a gamma counter (Cobra II Auto-
Gamma Counter; Packard Biosciences Co.). The remaining
cell lysate was used post radioactive decay for determination
of protein concentration using a bicinchoninic acid (BCA)
assay (Thermo Fisher Scientific Inc.). Additionally, 10 μl
standards from the solution of [18F]DASA-23 in DMEM
(25 μCi/ml) were counted to quantitate the percentage of
radiotracer uptake. For efflux studies, HeLa cells were
incubated with [18F]DASA-23 for 60 min and then washed
with room temperature Hanks’ buffered salt solution
(3 × 500 μl); a subsequent incubation was then completed
at 37 °C in fresh, prewarmed radiotracer-free DMEM. At the
specified times, samples were processed as described above.
Results
Chemistry
Scheme 1 illustrates the synthesis of 1-((2-fluoro-6-
nitrophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine
(10), the [18F]DASA-23 precursor. The synthesis begins with
reaction of commercially available 4-methoxybenzenesulfonyl
chloride with tert-butyl piperazine-1-carboxylate to afford inter-
mediate compound 8. Cleavage of the boc-protecting group and
subsequent reaction with 3-fluoro-5-nitrobenzenesulfonyl chlo-
ride afford the desired precursor 10 in 36 % yield over three steps.
Scheme 2 describes the synthesis of cold DASA-23 standard
which is readily prepared via reaction of previously synthesized
intermediate compound 8 with 3,5-difluorobenzenesulfonyl
chloride.
Radiochemistry
[18F]DASA-23 (1) (Scheme 3) was prepared by nucleophilic
displacement of the nitro group within 10 by the [18F]fluo-
ride ion in DMF at 110 °C for 20 min. Subsequent
purification by semipreparative HPLC yielded pure
In Vitro Serum Stability Studies
[
1 8 F]DASA-23. The radiochemical yield was
Human and mouse serum (Sigma-Aldrich) were centrifuged
at 4 °C, with speed of 13,000 rpm for 10 min. Formulated
2.61 1.54 %, non-decay corrected at EOS, and the specific
activity was 2.59 0.44 Ci/μmol (n = 10). The identity of
Scheme 3. Synthesis of [18F]DASA-23. Reagents and conditions. a [18F]KF/K2.2.2, DMF, 110 °C, 20 min, 2.61 1.55 % non-
decay corrected at EOS (n = 10).

Beinat C. et al.: Development of [18F]DASA-23
Fig. 2. Analytical HPLC profile of co-injection of a [18F]DASA-23 with b DASA-23 standard
[18F]DASA-23 was confirmed by analytical HPLC co-
injection with cold standard (Fig. 2).
[18F]DASA-23 was 95.7 and 95.8 for respective incubation
with human and mouse serum (Fig. 4).
Cell Uptake Studies
Discussion
Clinically relevant imaging agents for the direct, noninvasive
imaging of PKM2 are a topic of great interest and
importance. Molecular imaging with PET of PKM2 expres-
sion has the potential to play an extremely important role in
the noninvasive detection and measurement of malignancies
where [18F]FDG fails. The present work describes the
synthesis and radiolabeling of [18F]DASA-23, a PKM2
activator from the N-N′-diarylsulfonamide class of ligands,
combined with its preliminary in vitro characterization using
cell uptake and stability studies.
DASA-23 was identified as an appropriate compound for
radiolabeling due to its potency as a PKM2 activator, with a
maximum activating concentration (AC50) of 90 nM and
also its selectivity for PKM2 relative to other PK enzymes,
PKM1, PKR, and PKL [14]. This class of PKM2 activator
has been described to activate the PKM2 isoform by
reducing the KM (Michaelis constant) for PEP while
conserving the affinity of PKM2 for ADP [14]. PKM2
activators are known to bind a pocket at the PKM2 subunit
interface which promotes association of PKM2 subunits into
Cell culture studies showed rapid cellular uptake of the
tracer in HeLa tumor cells post addition of [18F]DASA-23,
with uptake values of 11.4 2.2 and 21.0 2.0 % of total
tracer/mg protein at 30 and 60 min, respectively (Fig. 3).
Removal of tracer from the cell media resulted in some
efflux of the cell-associated radioactivity with 52.8 4.8 %
of tracer still retained 30 min after removal of the
[18F]DASA-23 from media. Upon addition of TEPP-46, a
structurally different class of PKM2 activator, which binds
to the same allosteric site as the DASA-23 class of
compounds, significant blocking (70 %, p G 0.01) of tracer
uptake was observed (Fig. 3).
Stability Studies
In vitro plasma stability studies showed that [18F]DASA-23
was stable following incubation with either human or mouse
serum at 37 °C for up to 120 min. The percentage of intact
Fig. 3. Tumor cell uptake, retention, and washout of [18F]DASA-23. a Cell uptake. b Retention of [18F]DASA-23 was assessed
in human HeLa cervical adenocarcinoma cells. c Specificity of [18F]DASA-23 uptake in HeLa cells was also assessed by co-
incubating cells with TEPP-46 for 30 min. Data are means SD (n = 2 separate experiments, samples were run in triplicates for
each experiment). **p G 0.01.

Beinat C. et al.: Development of [18F]DASA-23
Fig. 4. In vitro human and mouse serum stability of [18F]DASA-23. a Percentage of intact tracer over time. b Representative
analytical HPLC profiles at t = 120 min, [18F]DASA-23 (peak 1) and unknown metabolite (peak 2).
stable tetramers [18]. Structural analyses of activators bound
to PKM2 tetramers have revealed a binding pocket present at
the interface of the subunit interaction, which was distinct
from the FBP binding site. Small molecular activators were
found to stabilize the A-A′ interface of the active tetramer,
unlike FBP which stabilizes the C-C′ interface [18]. No
natural ligands have been identified for this binding site of
PKM2, considering that multiple classes of activators bind
this pocket;it is possible that this signifies a novel site of
pyruvate kinase regulation.
110 °C for 20 min (precursor concentration 300 μg/ml); this
afforded high purity product in sufficient yields
(2.61
1.54 %) for preliminary in vitro characterization.
Since heating the reaction at 110 °C for 20 min afforded
high purity product in sufficient yields for preliminary
in vitro evaluation, no further optimizations were pursued
at this stage. Future efforts will focus on improving the
radiochemical yield with potential areas of exploration being
shorter reaction times and optimization of reaction temper-
ature. Our evaluation of the tracer in HeLa cells showed that
[18F]DASA-23 exhibited rapid and high levels of uptake and
retention. Rapid uptake is necessary for [18F]DASA-23, with
a half-life of 110 min, to be an effective PET tracer. The
specificity of this tracer for PKM2 was confirmed by co-
incubating cells with [18F]DASA-23 and TEPP-46. TEPP-46
is a structurally distinct PKM2 activator (AC50 = 92 nM)
known to bind the same allosteric pocket as DASA-23,
therefore blocking the PKM2 activator binding. This
resulted in significant blocking of tracer uptake, therefore
indicating that the tracer is specific and may be a suitable
marker of PKM2 expression levels. In vitro stability studies
revealed that [18F]DASA-23 is stable in both human and
mouse serum up to 120 min. Although this suggests that
[18F]DASA-23 may be transported to tissues in its intact
form, further in vivo metabolism studies are required to
confirm this. Taken together, these data highlight the
potential of [18F]DASA-23 as a specific radiotracer for the
precise identification and visualization of PKM2 expression.
We have previously demonstrated that [11C]DASA-23
successfully detects orthotopically growing human glioblas-
toma with aberrantly expressed PKM2 in mouse models; the
intrinsically low background of PKM2 in the healthy brain
also provided an optimal signal-to-background ratio [17].
The in vivo specificity of [11C]DASA-23 for PKM2 was
demonstrated through competition studies with structurally
distinct PKM2 activator, TEPP-46 [17]. This work sets the
foundation for clinical translation of [11C]DASA-23 for the
imaging of primary brain tumors and potentially other
tumors that metastasize to the brain. The favorable physical
characteristics of fluorine-18 (t1/2 = 110 min) relative to
carbon-11 (t1/2 = 20.4 min) and the presence of fluorine
atoms in the native structure of DASA-23 prompted us to
introduce the F-18 label into the native position to facilitate
clinical translation of this radiotracer. Labeling DASA-23
with F-18 in the native site on the molecule will not alter in
any way the compound structure and therefore already
established binding to PKM2.
Radiolabeling of DASA-23 was achieved by fluorine-18
nucleophilic displacement of the aryl nitro moiety within
compound 10. The nucleophilic displacement by fluorine-18
of a nitro group activated by electron-withdrawing groups is
a well-established and efficient method for the preparation of
no carrier-added F-18-labeled aromatics [19–21]. This
methodology has been widely applied to the preparation of
radiopharmaceuticals (a notable example being
[18F]spiroperidol) and for the preparation of synthetically
useful labeled intermediates [20]. Radiolabeling of DASA-
23 was achieved by heating the fluorination reaction at
Conclusion
Herein, we have identified an F-18-labeled PKM2-specific
radiotracer which shows potential for in vivo imaging.
Preliminary cell uptake and stability studies support the
application of this radiotracer for the detection of disease in
which PKM2 expression is relevant. In future studies, we
will further examine the tracer uptake in other cell lines such
as human glioblastoma lines and determine the efficacy of
this radioligand in imaging PKM2 expression in various
murine tumor models. These data set a foundation for

Beinat C. et al.: Development of [18F]DASA-23
exploring the utility of [18F]DASA-23 as a PKM2-specific
imaging agent in the context of human disease.
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functions of the key glycolytic regulator PKM2 in cancer. Trends
Biochem Sci 37:309–316
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kinase. The effect of preincubation, pH, copper ions, fructose 1,6-
diphosphate and dietary changes on enzyme activity. Biochem J
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M2 is a phosphotyrosine-binding protein. Nature 452:181–186
12. Bluemlein K, Grüning N-M, Feichtinger RG et al (2011) No evidence
for a shift in pyruvate kinase PKM1 to PKM2 expression during
tumorigenesis. Oncotarget. doi:10.18632/oncotarget.278
Acknowledgements. We would like to thank funding support from the Ben
and Catherine Ivy Foundation. We would also like to thank the
Radiochemistry Facility at Stanford University for the 18F-18 production
and Dr. Bin Shen and Jun Hyung Park for the helpful discussions about this
synthesis.
Compliance with Ethical Standards
Conflict of Interest
13. Luo W, Semenza GL (2012) Emerging roles of PKM2 in cell
metabolism and cancer progression. Trends Endocrinol Metab
23:560–566
The authors declare that they have no conflict of interest.
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